Malaria is the world's most important parasitic infectious disease, in terms of the number of exposed and infected individuals. It causes an estimated 2-3 million deaths per year, primarily among children under 5 years old in Africa, and constitutes a heavy economic burden in affected communities. Among the four species of Plasmodium responsible for human malaria, Plasmodium falciparum causes by far the most morbidity and mortality. The rapidly-increasing incidence of drug-resistant parasite strains, as well as insecticide-resistant mosquito vectors, have drastically increased the urgency of producing an effective vaccine to combat this wide spread plague of poverty. The asexual blood stage of the Plasmodium life cycle is responsible for the clinical symptoms and pathology of malaria. The extra-cellular merozoite is the infectious form of the parasite for red blood cells, and its surface proteins are obvious vaccine candidates, since many are likely to play essential roles in the erythrocyte invasion process, and they are accessible targets for humoral immune system effectors (antibodies and complement).
MSP-1 has already been the subject of a number of studies. It is synthesized in the schizont stage of Plasmodium type parasites, in particular Plasmodium falciparum, and is expressed in the form of one of the major surface constituents of merozoites both in the hepatic stage and in the erythrocytic stage of malaria (1, 2, 3, 4). Because of the protein's predominant character and conservation in all known Plasmodium species, it has been suggested that it could be a candidate for constituting anti-malarial vaccines (5, 6).
The same is true for fragments of that protein, particularly the natural cleavage products which are observed to form, for example during invasion by the parasite into erythrocytes of the infected host. Among such cleavage products are the C-terminal fragment with a molecular weight of 42 kDa (7, 8) which is itself cleaved once more into an N-terminal fragment with a conventional apparent molecular weight of 33 kDa and into a C-terminal fragment with a conventional apparent molecular weight of 19 kDa (9) which remains normally fixed to the parasite membrane after the modifications carried out on it, via glycosylphosphatidylinositol (GPI) groups (10, 11).
It is also found at the early ring stage of the intraerythrocytic development cycle (15, 16), whereby the observation was made that the 19 kDa fragment could play a role which is not yet known, but which is doubtless essential in re-invasive processes. This formed the basis for hypotheses formed in the past that that protein could constitute a particularly effective target for possible vaccines.
Among the most promising blood stage vaccine candidates is the major merozoite surface protein 1 (MSP1), which has been studied most extensively in Plasmodium falciparum, although homologues exist for all other Plasmodium species studied, such as for Plasmodium vivax for example. It is synthesized during schizogony as a 185-215 kDa protein (depending on the species), which is attached to the merozoite plasma membrane by a C-terminal glycosyl-phosphatidyl-inositol (GPI) moiety [1]. During merozoite maturation. MSP1 undergoes two successive proteolytic cleavages, giving rise to an 11 kDa C-terminal peptide, known as MSP1p19 due to its migration on SDS-PAGE. MSP1p19 with its GPI anchor is the only part of MSP1 remaining on the merozoite in newly invaded erythrocytes [2] reviewed in [3]. The precise function(s) of MSP1 and/or MSP1p19, although unknown, appear to be essential for erythrocyte invasion and parasite survival, since no viable parasites can be recovered following MSP1 knock-out, although surprisingly. PfMSP1p19 can be complemented by its P. chabaudi homologue [4]. Unlike many polymorphic Plasmodium merozoite surface proteins [5], PfMSP1p19 is highly conserved in parasites from diverse geographical locations [6]; [7]; [8]; [9], a feature with obvious advantages for a vaccine candidate. MSP1p19 in all species is highly cysteine rich and is composed essentially of two tandem, closely associated epidermal growth factor-like domains. Although the amino acid sequence of MSP1p19 varies considerably among different Plasmodium species, its 3-dimentional structure appears to be remarkably conserved [10]; [11]. Indeed its function appears to depend exclusively on its structure than to any given amino acid sequence [4].
It should be understood that the references frequently made below to the p42 and p19 proteins from a certain type of Plasmodium are understood to refer to the corresponding C-terminal cleavage products of the MSP-1 protein of that Plasmodium or, by extension, to products containing substantially the same amino acid sequences, obtained by genetic recombination or by chemical synthesis using conventional techniques, for example using the “Applied System” synthesizer, or by “Merrifield” type solid phase synthesis. For convenience, references to “recombinant p42” and “recombinant p19” refer to “p42” and “p19” obtained by techniques comprising at least one genetic engineering step.
Faced with the difficulty of obtaining large quantities of parasites for P. falciparum and the impossibility of cultivating P. vivax in vitro, it has become clear that the only means of producing an anti-malaria vaccine is to resort to techniques which use recombinant proteins or peptides. However. MSP-1 is very difficult to produce whole because of it large size of about 200 kDa, a fact which has led researchers to study the C-terminal portion, the (still unknown) function of which is probably the more important.
Recombinant proteins concerning the C-terminal portion of the P. falciparum MSP-1 which have been produced and tested in the monkey (12, 40, 41) are:                a p19 fused with a glutathione-S-transferase produced in E. coli (40);        a p42 fused with a glutathione-S-transferase produced in E. coli (12);        a p19 fused with a polypeptide from a tetanic anatoxin and carrying auxiliary T cell epitopes produced in S. cerevisiae (12);        a p42 produced in a baculovirus system (41).        
A composition containing a p19 protein fused with a glutathione-S-transferase produced in E. coli combined with alum or liposomes did not exhibit a protective effect in any of six vaccinated Aotus nancymai monkeys (40).
A composition containing a p42 protein fused with a glutathione-S-transferase produced in E. coli combined with Freunds complete adjuvant did not exhibit a protective effect in two types of Aotus monkeys (A. nancymai and A. vociferans) when administered to them. The p19 protein produced in S. cerevisiae exhibited a protective effect in two A. nancymai type Aotus monkeys (12). In contrast, there was no protective effect in two A. vociferans type Aotus monkeys.
Some researchers (18) have also reported immunization tests carried out in the rabbit using a recombinant p42 protein produced in a baculovirus system and containing one amino acid sequence in common with P. falciparum (18). Thus these latter authors indicate that in the rabbit that recombinant p42 behaves substantially in the same way as the entire recombinant MSP-1 protein (gp195). This p42 protein in combination with Freunds complete adjuvant has been the subject matter of a vaccination test in a non-human primate susceptible to infection by P. falciparum, Aotus, lemurinus grisemembra (40). The results showed that 2 of 3 animals were completely protected and the third, while exhibiting a parasitemia which resembled that of the controls, had a longer latent period. It is nevertheless risky to conclude to a protective nature in man of the antibodies thus induced against the parasites themselves. It should be remembered that there are currently no very satisfactory experimental models in the primate for P. vivax and P. falciparum. The Saimiri model, developed for P. falciparum and P. vivax, and the Aotus model for P. falciparum, are artificial systems requiring the parasite strains to be adapted and often requiring splenectomy of the animals to obtain significant parasitemia. As a result, the vaccination results from such models can only have a limited predictive value for man.
In any event, what the real vaccination rate would be which could possibly be obtained with such recombinant proteins is also questionable, bearing in mind the discovery—reported below—of the presence in p42s from Plasmodiums of the same species, and more particularly in the corresponding p33s, of hypervariable regions which would in many cases render uncertain the immunoprotective efficacy of antibodies induced in individuals vaccinated with a p42 from a Plasmodium strain against an infection by other strains of the same species (13).
It can even be assumed that the high polymorphism of the N-terminal portion of p42 plays a significant role in immune evasion, often observed for that type of parasite.
The aim of the invention is to produce vaccinating recombinant proteins which can escape these difficulties, the protective effect of which is verifiable in genuinely significant experimental models or is even directly in man.
More particularly, the invention provides vaccinating compositions against a Plasmodium type parasite which is infectious for man, containing as an active principle a recombinant protein which may or may not be glycosylated, whose essential constituent polypeptide sequence is:                either that of a 19 kilodalton (p19) C-terminal fragment of the surface protein 1 of the merozoite form {MSP-1 protein) of a Plasmodium type parasite which is infectious for man, said C-terminal fragment remaining normally anchored to the parasite surface at the and of its penetration phase into human erythrocytes in the event of an infectious cycle;        or that of a portion of that fragment which is also capable of inducing an immune response which can inhibit in vivo parasitemia due to the corresponding parasite;        or that of an immunologically equivalent peptide of said p19 fragment or said portion of that fragment; and said recombinant protein further comprises conformational epitopes which are unstable in a reducing medium and which constitute the majority of the epitopes recognized by human antiserums formed against the corresponding Plasmodium.        
The presence of such conformational epitopes plays an important role in the protective efficacy of the active principle of the vaccines. They are particularly found in the active principles which exhibit the other characteristics defined above, when they are produced in a baculovirus system. If need be, it is mentioned below that the expression “baculovirus vector system” means the ensemble constituted by the baculovirus type vector itself and the cell lines, in particular cells of insects transfectable by a baculovirus modified by a sequence to be transferred to these cell lines resulting in expression of that transferred sequence. Preferred examples of these two partners in the baculovirus system have been described in the article by Longacre et al. (14). The same system was used in the examples below. It goes without saying, of course, that variations in the baculovirus and in the cells which can be infected by the baculovirus can be used in place of those selected.
In particular, the recombinant protein is recognized by human antiserums formed against the corresponding Plasmodium or against a homologous Plasmodium when it is in its non reduced state or in a reduced non irreversible state, but is not recognized or is only recognized to a slight extent by these same antiserums when it is irreversibly reduced.
The unstable character of these conformational epitopes in a reducing medium can be demonstrated by the test described below in the examples, in particular in the presence of β-mercaptoethanol. Similarly, the examples below describe the experimental conditions applicable to obtain irreversible reduction of the proteins of the invention.
From this viewpoint, the recombinant protein produced by Longacre et al. (14) can be used in such compositions. It should be remembered that S. Longacre et al. succeeded in producing a recombinant p19 from the MSP-1 of P. vivax in a baculovirus vector system containing a nucleotide sequence coding for the p19 of Plasmodium vivax, in particular by transfecting cultures of insect cells [Spodoptera frugiperda (Sf9) line] with baculovirus vectors containing, under the control of the polyhedrin promoter, a sequence coding for the peptide sequences defined below, with the sequences being placed in the following order in the baculovirus vector used:                a 35 base pair 5′ terminal fragment of the polyhedrin signal sequence, in which the methionine codon for initiating expression of this protein had been mutated (to ATT);        a 5′-terminal nucleotide fragment coding for a 32 amino acid peptide corresponding to the N-terminal portion of MSP-1, including the MSP-1 signal peptide;        either a nucleotide sequence coding for p19, or a sequence coding for the p42 of the MSP-1 protein of Plasmodium vivax, depending on the case, these sequences also being provided with (“anchored” forms) or deprived of (soluble forms) 3′ end regions of these nucleotide sequences, whose end C-terminal expression products are reputed to play an essential role in anchoring the final p19 protein to the parasite membrane;        2 TAA stop codons.        
For p42, the sequences derived from the C-terminal region of MSP-1 extend consequently from amino acid Asp 1325 to amino acid Leu 1726 (anchored form) or to amino acid Ser 1705 (soluble form) and for p19, the sequences extend from amino acid Ile 1602 to amino acid Leu 1726 (anchored form) or to amino acid Ser 1705 (soluble form) it being understood that the complete amino acid sequences of p42 and p19, whose initial and terminal amino acids have been indicated above follow from the gene of the Belem isolate of P. vivax which has been sequenced (20).
In studies carried out in endemic regions, PfMSP1p19 specific antibodies in the sera of exposed individuals have been associated with protection against high levels of parasitemia and clinical episodes [12]; [13]; [14]; [15]; [16]; [17]. In addition, anti-MSP1 antibodies in human immune sera have been shown to inhibit parasite growth in vitro [18]. More importantly, using transfected P. falciparum and P. chabaudi parasite lines with reciprocal allelic replacements of their divergent MSP1p19 gene sequences (43% homology), O'Donnell et al. [19] could show that antibodies specific for this domain in P. falciparum immune human sera or P. chabaudi immune mice, represented a major component of the growth inhibition, or invasion inhibitory responses in parasite cultures.
Several vaccination trials have been carried out in primate models using varying recombinant versions of C-terminal MSP1 (p42 and/or p19) in association with a number of different adjuvants, which have provided some indication of parameters that may be important for protective efficacy. None of the C-terminal PfMSP1 recombinant proteins expressed in E. coli have shown evidence of protective efficacy in primates [20]; [21]; [22]). Protective efficacy conferred by yeast recombinant versions of PfMSP1p19 has been highly inconsistent, depending on the subspecies of Aotus used [20], the challenge dose of infected red blood cells [23], and the exact nature of the recombinant construct [22]; [24]. (In contrast, recombinant C-terminal MSP1 proteins produced in the baculovirus expression system have consistently shown good protective efficacy in primates [25]; [26]; [27].)
Many parasitic protozoa with extra-cellular phases in their life cycles, such as Plasmodium, appear to express a majority of their surface proteins such that a C-terminal hydrophobic signalling sequence has been replaced with GPI moieties [28]. The primary function of GPI structures is to mediate a stable association of surface proteins to the plasma membrane, such that the functional ectoplasmic domain faces the external environment, in relative isolation with intracellular components. In particular, several P. falciparum merozoite surface proteins have GPI anchors, including MSP1, MSP2, MSP4 and MSP5 [28], the structure of which has been determined for MSP1 and MSP2 [1]. Recently P. falciparum GPIs have been implicated in the pathology of clinical malaria disease by inducing TNFα secretion, leading to fever and the up-regulation of capillary epithelial adhesion molecules that can bind infected erythrocytes causing capillary obstruction and associated pathologies [29]; [30]; [31]; [32, 33]; [34]. Indeed, individuals who live in malaria endemic areas can develop a specific anti-P. falciparum GPI antibody response, which is correlated with age and, in some cases, protection against disease [34]; [35]; [36]. In addition, mice immunized with synthetic P. falciparum GPI showed enhanced survival and reduced P. berghei induced symptoms of malaria. Moreover, antibody directed against this synthetic GPI could neutralize in vitro pro-inflammatory activity by P. falciparum [37].
In humans, immune responses to MSP1p19 as well as to P. falciparum GPIs have been associated with protection against clinical malaria [12]; [13]; [34]. The inventors were interested in developing means that can be encompassed in a vaccine candidate, which could permit to elicit an anti-toxic as well as an antiparasitic immunity, which could have an improved efficiency with respect to the immune response observed in various models, against soluble baculovirus MSP1p19.
Martinez et al [53] expressed a “recombinant ookinete surface antigen Pbs21 of Plasmodium berghei when prepared in a baculovirus SP21 insect cell expression system>>. The Pbs21 antigen expressed in the baculovirus system was disclosed by Martinez et al, to be produced in the cytoplasm of Sf21 cells, on their surface and in the extracellular medium. The expressed antigen was affinity purified starting from Sf21 cells resuspended in a solubilization buffer. Immunogenicity of the purified recombinant antigen was tested in mice and compared with immunogenicity of the native Pbs21 protein. The recombinant antigen was found to be less immunogenic than the native Pbs21. When discussing the obtained results. Martinez et al reported that recombinant Pbs21 (1-213) bearing the Spodoptera GPI anchor was less immunogenic but that the data obtained would suggest that the conformation and the presence of a GPI anchor is critical to the immunogenicity. It is furthermore disclosed that the majority of the response induced is to the folded polypeptide and not its post-translational additions.
However, although, several GPI-anchored proteins have already been expressed in an expression system, the capacity of expression systems to reproduce this anchor for the Plasmodium merozoite surface protein MSP1 remains controversial.
The invention relates to the objects defined in the claims, and more particularly to:                purified polypeptides comprising or essentially consisting of a C-terminus MSP1 antigen from Plasmodium carrying a Glycosyl-phosphatidyl-inositol group (GPI),        purified variants having at least 80% identity with such polypeptides,        immunogenic compositions, wherein said composition comprises or essentially consists of at least one purified polypeptide or variant of the invention, and a pharmaceutically acceptable vehicule,        vaccines against Plasmodium infection, wherein said vaccine comprises or essentially consists of at least one active principle, which is a purified polypeptide or variant of the invention,        nucleic acid molecules, which encode the amino acid sequence of a purified polypeptide of the invention, or the amino acid sequence of a variant of the invention,        nucleic acid molecules, which encode the amino acid sequence of a purified polypeptide of the invention, or the amino acid sequence of a variant of the invention, which is linked to at least one nucleic acid molecule, which encodes a signal peptide essentially consisting of, or comprising, the following sequence MKIIFFLCSFLFFIINTQC (SEQ ID NO: 3) and at least one nucleic acid molecule, which encodes at least one anchor peptide, which signals for GPI addition, said anchor peptide essentially consisting of or comprising the following sequence SSNFLGISFLLILMLILYSFI (SEQ ID NO: 4),        nucleic acid molecules, which encode a purified polypeptide of the invention or a variant of the invention, which has a nucleotide sequence, which is optimised for codon usage appropriate in baculovirus expression vector,        expression vectors for the expression of at least one purified polypeptide the invention, comprising at least of said nucleic acid molecules of the invention,        expression system for the expression of at least one purified polypeptide of the invention, or of a variant of the invention, wherein said expression system comprises a baculovirus expression vector of the invention and insect cells,        the recombinant baculovirus PfMSP1p19/His/GPI deposited at the C.N.C.M. on Nov. 10, 2005 under the accession number CNCM I-3515,        the recombinant baculovirus PvMSP1p19/His/GPI deposited at the C.N.C.M. on Nov. 10, 2005 under the accession number CNCM I-3516,        a polynucleotide, which essentially consists of the nucleic acid fragment of MSP1p19 contained in PfMSP1p19/His/GPI (CNCM I-3515) or in PvMSP1p19/His/GPI (CNCM I-3516).        